WO2012038918A1 - Therapeutic product inhibitor of the cell proliferation and biological applications thereof - Google Patents

Abstract

Arpp19 and/or α-Endosulfine for use as specific inhibitors of the cell cycle deregulation and biological applications thereof.

Description

Therapeutic product inhibitor of the cell proliferation and biological applications thereof

The invention relates to specific inhibitors of the cell cycle deregulation and the biological applications thereof.

Mitotic entry and exit were historically thought to be directly dependent on the activation and inactivation of the cyclin B-Cdc2 complex. Recent results have expanded this model to include phosphatases. New data indicates that maintenance of phosphorylations on mitotic proteins is not exclusively dependent on cyclin B- Cdc2 kinase, but also on the activity of the phosphatase PP2A, which is the main phosphatase responsible for the dephosphorylation of mitotic substrates although a role of PP1 has also been reported in Xenopus egg extracts. Therefore the balance between cyclin B-Cdc2 and PP2A activities determines the timing of mitotic entry and exit.

The mechanisms controlling cyclin B-Cdc2 activity have been largely described whereas very little is known about the mechanisms controlling PP2A activity during mitosis. In this regard, three studies have recently demonstrated a role of the Greatwall kinase in the regulation of PP2A activity during mitosis. These works demonstrate that the depletion of Gwl from mitotic egg extracts induces mitotic exit, whereas the depletion of this protein from interphase egg extracts prevents mitotic entry. Moreover the authors show that these phenotypes, as well as the phenotypes observed in Gwl knockdown human cells, are mediated by PP2A. PP2A regulation might then be mediated by phosphorylation, however, while Gwl and PP2A can interact, a direct phosphorylation on PP2A by this kinase, has never been observed suggesting that Gwl inhibits PP2A through an intermediary protein.

In the framework of the invention, the inventors used biochemical fractionation of Xenopus egg extracts and in vitro phosphorylation with a hyperactive form of human Gwl to identify the first substrates of this kinase.

Interphase Xenopus egg extracts were first fractionated with a heparin column. The elution of this column was subsequently submitted to ammonium sulphate precipitation (cut off 50-70%) and finally resuspended and fractionated by Gel Filtration. The different fractions were subsequently phosphorylated in vitro with a hyperactive form of human Gwl (K72M mutant) (7) and the phosphorylated bands were analysed by mass spectrometry. Maximal phosphorylation was observed in the Gel Filtration fractions corresponding to molecular weights of 30 to 70 (Figure 1A, Fraction 3) and 20 to 50 kDa (Figure 1A, Fraction 4). These two fractions presented a clear phosphorylated band of a molecular weight of around 20 kDa. This band was cut and analysed by mass spectrometry. Forty different proteins were identified, among them the cAMP-regulated phosphoprotein 19 (Arppl9), a protein that strongly resembles the small protein a-Endosulfine. The role of Arppl9 and a- Endosulfine are unclear. Arppl9 was found to be selectively enriched in neostriatum but no clear biological role has been assigned (2), while a-Endosulfine was first suggested to be involved in the control of insulin release from pancreatic β cells through the regulation of ATP-dependent potassium channels, however this hypothesis appears to be no longer supported. However, recent data indicates that oocytes from Drosophila with mutant a-Endosulfine have a prolonged prophase and fail to progress to metaphase. Moreover these oocytes contain normal mitotic levels of in vitro Cdc2 kinase activity, but importantly show a reduced amount of in vivo phosphorylation on mitotic substrates, a phenotype reminiscent to the one observed in Gwl-depleted Xenopus egg extracts.

Although by mass spectrometry peptides specific to Arppl9 but not for cc- Endosulfme (Supplementary data, Figure 1) were identified, it could not be excluded that both of these proteins were putative Gwl substrates due to their close sequence homology.

Furthermore, as the a-Endosulfine phenotype observed in Drosophila oocytes was very similar to the one observed in Gwl-depleted mitotic extracts, the inventors decided to focus on both Arppl9 and a-Endosulfine, as possible putative substrates of Gwl.

The inventors results show that both proteins were clearly phosphorylated in vitro by the Gwl kinase (Figure IB). However, the endogenous proteins were present at low levels in Xenopus egg extracts, especially Arppl9, that was very difficult to detect unless an immunoprecipitation from a large excess of extracts was used and a pre- boiling treatment of this extract was performed before Laemmli buffer addition to eliminate the non-thermo stable unspecific bands (Figure 1C).

The analysis of the protein sequence of Arppl9 (h Arpp 19 and x Arpp 19 having SEQID N° 1 and SEQID N° 2, respectively) and CC-Endosulfme (of sequence SEQID N° 3) revealed the presence of seven potential serine/threonine phosphorylation sites conserved in both proteins (Figure ID).

A directed mutagenesis of the human Arpp 19 protein to obtain individual S/T to A mutants of this protein was performed and then tests were carried out to know whether they were phosphorylated in vitro by Gwl.

All mutants, including the PKA phosphorylation site S104A, were phosphorylated by Gwl except mutant S62A. Similarly, mutation of this conserved serine of human CC- Endosulfme to alanine (S67A) also prevented the phosphorylation of this protein by GW (Figure ID).

These results indicate this serine is the Gwl phosphorylation site in both Arpp 19 and a-Endosulfme.

The effect of the addition of Arpp 19 and α-Endosulfme to interphase Xenopus egg extracts was next analysed.

The addition of these two proteins, previously phosphorylated in vitro by Gwl, promoted a rapid mitotic entry as shown by the phosphorylation of the mitotic substrate Gwl and by the activation of cyclin B-Cdc2 (Figure IE, note dephosphorylation of tyrosine 15 of Cdc2), followed by a subsequent exit of mitosis as indicated by the degradation of cyclin A and cyclin B2. This mitotic entry and exit presented similar kinetics to the first mitotic division of cycling egg extracts.

In a second experiment, to easily visualize mitotic entry, cyclin degradation was prevented by depleting the Anaphase Promoting Complex constituent Cdc27.

In these extracts, the addition of either Arpp 19 or α-Endosulfme again promoted a rapid entry into mitosis and a maintenance of the mitotic state, but only when these two proteins were previously phosphorylated by Gwl, and not when the non- phosphorylated or when the Arpp 19 S62A or the a-Endosulfme S67A phosphorylation mutants were used (Figure 2A).

Two different activities, cyclin B-Cdc2 and PP2A, must be regulated to promote mitotic entry. It is known that Gwl mediates entry into mitosis through the inhibition of PP2A, and thus, phosphorylated Arppl9 and α-Endosulfme probably mediate mitotic entry by directly inhibiting PP2A. However, the possibility that these two proteins could also regulate cyclin B-Cdc2 activity to promote entry into mitosis cannot be excluded.

The inventors have previously demonstrated that when cyclin B-Cdc2 activation is prevented in interphase egg extracts by Cdc25 depletion, the inhibition of PP2A induces entry into mitosis although only a partial phosphorylation of mitotic substrates by cyclin A-Cdc2 is observed (3).

To test whether Arppl9 and α-Endosulfme could induce mitotic entry through the regulation of cyclin B-Cdc2 activity, the activation of cyclin B-Cdc2 in interphase egg extracts was prevented by the depletion of Cdc25 and analysis was carried out to study whether the addition of these two proteins were still capable of inducing mitotic entry. Cdc27 was depleted to prevent cyclin degradation and to easily visualize mitotic entry in these extracts. As shown in Figure 2B, the addition of either phospho-Arppl9 or phospho-a-Endosulfme induced entry into mitosis as indicated by a decrease of the electrophoretic mobility of Gwl as well as an increase of the phospho-serine Cdk consensus motif staining in the absence of an active cyclin B-Cdc2 kinase (see pSerCdk Subst and phosphorylation of Tyrl5 of Cdc2 respectively).

However, as expected only cyclin A-Cdc2 was active since total HI kinase activity was low and the phosphorylation of Gwl was less prominent than in control mitotic extracts in which both cyclin A-Cdc2 and cyclin B-Cdc2 are active.

Thus, Arppl9 and α-Endosulfme are the downstream substrates of Gwl that promote mitotic entry, likely through the regulation of PP2A, but not through the regulation of cyclin B-Cdc2.

Accordingly, when Gwl was depleted from interphase egg extracts the addition of cc- Endosulfme and Arppl9 still induced mitotic entry in the absence of this kinase further demonstrating that they act downstream of Gwl (Figure 2C). It is worth noticing that these two proteins are only capable of promoting entry into mitosis in Gwl-depleted interphase extracts when they are thiophosphorylated, indicating that when Gwl is present, dephosphorylation of these two substrates is counterbalanced by endogenous Gwl, whereas, when Gwl is absent, these two proteins are rapidly dephosphorylated and are incapable of promoting and maintaining the mitotic state. Moreover, the addition of thiophosphorylated Arppl9 or α-Endosulfme to Gwl- depleted CSF extracts also prevented mitotic exit (Figure 2D) demonstrating that Arppl9 and CC-Endosulfme are phosphorylated by Gwl and act downstream of this kinase to promote mitotic entry, possibly by directly inhibiting PP2A phosphatase. Accordingly, the phosphorylation of the Gwl-specific site on Arppl9 and CC- Endosulfme (S62/S67) disappeared simultaneously with dephosphorylation of Gwl at mitotic exit (Figure 2E) and it reappeared again concomitantly with the phosphorylation of this kinase at mitotic entry (Figure 2F).

Therefore the remaining question was to determine whether Arppl9 and CC- Endosulfme could bind and inhibit PP2A. To do that, a GST-pull down with GST- Arppl9 and GST-a-Endosulfme in interphase and in mitotic egg extracts was then performed.

As shown in Figure 3 A, GST-Arppl9 and GST-a-Endosulfme weakly bound both the PP2A A and C subunits in interphase egg extracts in which Gwl is inactive, however, the binding significantly increased in mitotic egg extracts in which Gwl is fully active, suggesting that the association of these two proteins to PP2A/A and PP2A/C is highly stimulated by their Gwl-dependent phosphorylation. This is further supported by the fact that the mutation to alanine of the Gwl phosphorylation site S62 in Arrpl9 and to S67 in α-Endosulfme prevented the binding of these two proteins to PP2A. However, this association appears not to be exclusively regulated by Gwl-dependent phosphorylation since the α-Endosulfme mutant D66A, is unable to bind PP2A although it is normally phosphorylated by Gwl at S67 residue (Figure 3B). Moreover, the addition of this mutant previously phosphorylated by Gwl into interphase extracts does not induce mitotic entry (Figure 3C) further demonstrating that the final role of Gwl is the inhibition of PP2A by promoting the binding of this phosphatase to their inhibitors, Arppl9 and a-Endosulfine.

Next the activity of PP2A was tested by assessing its ability to dephosphorylate the cyclin B-Cdc2 substrate cMos in the presence or in the absence of thio-phospho- Arppl9, thio-phospho-a-Endosulfme and thio-phospho-a-Endosulfme D66A. The results show that thio-phospho-Arp l9 and thio-phospho-a-Endosulfme significantly decreased c-Mos dephosphoryaltion by PP2A, whereas the thio- phospho-a-Endosulfme mutant D66A which is unable to bind PP2A, did not have any effect on the activity of this phosphatase, indicating that these two proteins can directly bind and inhibit PP2A (Figure 3D).

The physiological role of Arppl9 and α-Endosulfme upon mitotic entry and exit was then analysed. To investigate mitotic entry, interphase extracts were first depleted of Cdc27, subsequently depleted of Arppl9 or of CC-Endosulfme and finally supplemented with cyclin A. As shown in Figure 4A, the complete depletion of cc- Endosulfme (see Figure 1C) did not prevent mitotic entry, whereas depletion of Arppl9 completely inhibited entry into mitosis. Similarly, the depletion of CC- Endosulfme from CSF extracts did not have any effect, whereas these extracts rapidly exited mitosis after Arppl9 depletion (Figure 4B). Moreover, this exit was mediated by a re-activation of PP2A since the artificially inhibition of PP2A by the addition of okadaic acid in Arppl9-depleted CSF extracts enabled these extracts to re-enter mitosis (Figure 4D). Thus, despite the fact that both proteins Arppl9 and CC- Endosulfme can act as PP2A inhibitors, only Arppl9 appears to have a relevant role during mitotic entry and exit in Xenopus egg extracts. This could be the consequence of a preferential phosphorylation of Arppl9 by Gwl since endogenous Arppl9 appears to be two times more phosphorylated than α-Endosulfme during mitosis (Figure 4C). This is in complete accord with the fact that despite the presence of higher levels of endogenous CC-Endosulfme compared to endogenous Arppl9 in Xenopus egg extracts, only Arppl9 has been identified in our biochemical analysis as a substrate of Gwl.

Finally, an analysis was carried out to determine whether this mechanism was also conserved in human cells. To do that Arppl9 in HeLa cells by siR A were knockeddown and the capacity of the cells to enter into mitosis was analysed. Knockdown of Arppl9 reduced the number of mitotic cells by 50% compared to the scramble control suggesting that, as it occurs for Xenopus egg extracts, Arppl9 is required to promote mitotic entry in human cells (figure 4E).

The inventors thus demonstrate that Arppl9 and a-Endosulfine are two substrates of Greatwall whose phosphorylation promotes the inhibition of PP2A. Accordingly, the data thus obtained show that both proteins are phosphorylated in vitro by Greatwall. It is also demonstrated that the addition of either of these two phosphorylated proteins to interphase extracts induces mitotic entry independently of the activity of cyclin B-Cdc2 and that this addition also rescues the phenotype induced by Gwl depletion in both interphase and mitotic extracts. It is further demonstrated that these two proteins bind PP2A preferentially when they are phosphorylated by Gwl and that this binding promotes significant inhibition of PP2A. Finally, it is also demonstrated that, despite the fact that both, Arppl9 and a-Endosulfine can inhibit PP2A in Xenopus egg extracts, Arppl9 is the main substrate of Gwl responsible of PP2A inhibition at mitotic entry.

The results shown here clearly demonstrate that Arppl9 plays an essential role in mitosis. Consequently, it is likely that a miss-regulation of the PP2A-inhibitory activity of this protein could result in aberrant mitosis as it has been previously demonstrated for its upstream kinase Gwl. Therefore, a correct timing of phosphorylation-dephosphorylation of this protein should be essential for correct cell division.

Besides the important role of Arppl9, the above also shows data that other physiological pathways could be regulated by a-Endosulfme-dependent inhibition of PP2A. In this regard, the physiological role of a-Endosulfine has to be determined as well as under which conditions is this protein phosphorylated by Gwl.

To summarize mitotic entry and maintenance requires the activation of cyclin B- Cdc2 and the inhibition of PP2A, which respectively phosphorylates and dephosphorylates mitotic substrates. The inventors previously demonstrated that the Greatwall (Gwl) kinase is required to maintain mitosis through PP2A inhibition. Here they describe the mechanism by which Gwl activation results in this PP2A inhibition. They identified Arppl9 and a-Endosulfine as two substrates of Gwl that, when phosphorylated, associate and inhibit PP2A promoting mitotic entry. Conversely, in the absence of Gwl activity, these two proteins are dephosphorylated and lose their capacity to bind and inhibit PP2A. Sequencing and directed mutagenesis experiments enabled to identify the phosphorylation site of Gwl (site S62), identical on both proteins. Finally, they show that while both proteins can inhibit PP2A, Arppl9 is the main Gwl substrate responsible for PP2A inhibition at mitotic entry in Xenopus egg extracts.

Phosphorylations occur through cyclin B-CDC2 kinase and result in mitose activation. A specific subpopulation of PP2A phosphatases is involved in the dephosphorylations, i.e. PP2A-B555 isoform. It results in mitose inhibition. The invention thus relates to Arppl9 and/or α-Endosulfme for use as specific inhibitors of the cell deregulation.

It also relates to a therapeutic product inhibitor of cell proliferation through PP2A, more particularly PP2A-B555 pathway.

The invention more particularly relates to the use as inhibitors of PP2A Β55δ complex of Arppl9 and CC-Endosulfme. Said compounds are valuable tools for therapy and in vitro diagnostic of pathologies resulting from cell cycle deregulation such as cancer.

Pharmaceutical compositions comprising an efficient amount of Arppl9 and cc- Endosulfine in association with pharmaceutically acceptable carriers are also covered by the invention. During the production of the drugs, the active ingredients, used in therapeutically effective amounts are mixed with the pharmaceutically acceptable vehicles for the mode of administration chosen. These vehicles may be solids or liquids or gels.

The amount of active principle in the drugs will be easily determined by those skilled in the art in view of the pathology to be treated. The doses per dosage unit will be chosen depending on the condition and age of the patient. The invention also encompasses an in vitro diagnostic method of pathologies original due to a cell cycle deregulation such as cancer comprising the use of Arppl9 and cc- Endosulfine. It also relates to a method of inhibiting said complex using Arppl9 and cc- Endosulfine.

FIGURE LEGENDS

Figure 1. Addition of Arp l9 and cc-Endosulfine previously phosphorylated in vitro by Gwl to interphase extracts promotes mitotic entry. (A) Coomassie Blue staining and autoradiography of two fractions of gel filtration in which a band of 20 kDa was highly phosphorylated in vitro. Arrowheads show bands that were analysed by mass spectrometry. (B) In vitro phosphorylation of Gst-Arppl9 and Gst-cc- Endosulfine by Gwl. (C) Western blot performed with anti-full length human Arppl9 (two expositons) and anti-Cterminal Xenopus cc-Endosulfine antibodies showing equal amounts of interphase and supernatant (10 μΐ in Arppl9 IP and 2 μΐ in cc-Endosulfine IP) and an IP corresponding to 20 μΐ of interphase extract. To improve endogenous Arppl9 detection extract and supernatant were boiled and centrifuged before Laemmli buffer addition to eliminate non-thermo stable unspecific bands. * Non-specific bands (D) Protein sequence alignment of human and Xenopus Arppl9 and Xenopus cc-Endosulfine. The conserved S/T sites that were mutated to A are indicated. Coomassie blue staining showing the amount of MBP- Arppl9 and ΜΒΡ-α-Endosulfme used for each mutant to perform in vitro phosphorylation. (E) Interphase extracts (INT) were or not (Control) supplemented with phospho-a-Endosulfme or phospho-Arppl9 at a final concentration of 170 ng/μΐ. Mitotic entry was determined by analysing the phosphorylation of Gwl, the dephosphorylation of tyrosine 15 on Cdc2 and the levels of cyclin A and cyclin B2.

Figure 2. Addition of thiophosphorylated Arppl9 and cc-Endosulfine rescues the phenotype induced by Gwl depletion in interphase and mitotic egg extracts. (A)

Interphase extracts were depleted of Cdc27 and supplemented at a final concentration of 170 ng/μΐ with the proteins Gst-Arppl9 and Gst-a-Endosulfme phosphorylated or not in vitro by Gwl. The Gwl-phosphorylation mutants Arppl9- S62A and a-Endosulfine-S67A were also submittd to in vitro phosphorylation and supplemented to these extracts. (B) Mitotic entry was analysed in interphase extracts that were depleted of Cdc27 and Cdc25 and supplemented with phospho-Arppl9 and phospho-a-Endosulfme (final concentration 170 ng/μΐ). The phosphorylation of cyclin B-Cdc2 substrates was analysed by using an antibody recognising the phospho- Serine Cdk consensus motif. (C) Interphase extracts were depleted of Cdc27 and Gwl and subsequently supplemented with phosphorylated or thiophosphorylated Arppl9 and a-Endosulfine. (D) Mitotic extracts (CSF) were supplemented with thiophosphorylated Arppl9 or a-Endosulfine and subsequently depleted of Gwl. (E) Mitotic exit was induced in CSF extracts by the addition of Ca²⁺ and the levels of cyclin B2 as well as the phosphorylation of Cdc25, of Gwl and of the Gwl specific sites of Arppl9 and α-Endosulfine (S62 and S67 respectively) were analysed. (F) Interphase extracts were supplemented with cyclin A (final concentration 60 nM) and the kinetics of the phosphorylation of the indicated proteins as well as the levels of cyclin B2 were analysed upon mitotic entry.

Figure 3. Phosphorylated Arppl9 and α-Endosulfine bind and inhibit PP2A. (A) Interphase or mitotic extracts were supplemented with Gst-Sepharose, Gst-Arppl9- Sepharose, Gst-a-Endosulfme-Sepharose, the Gst-Arppl9 (S62A)-Sepharose or Gst- a-Endosulfme (S67A)-Sepharose and twenty minutes later the binding of PP2A subunits A and C were analysed by GST pull down and western blot. (B) Similar to (A) except for the addition of Gst-a-Endosulfme-Sepharose and GST-a-Endosulfme (D66A)-Sepharose to these extracts. Lower panel shows the in vitro phosphorylation of the α-Endosulfine (D66A) mutant by Gwl. (C) The capacity of the phospho-a- Endosulfme (D66A) mutant to induce mitotic entry in interphase extracts was analysed by measuring phosphorylation of Gwl and dephosphorylation of tyrosine 15 of Cdc2. (D) PP2A complex obtained by immunoprecipitation from 60 μΐ of interphase extracts was or not (CT) pre-incubated for 10 minutes with 5.3 μg of either thiophospho-Gst-Arpp 19, thiophospho-Gst-a-Endosulfme or thiophospho-Gst- a-Endosulfme (D66A) and subsequently mixed with phospho-radiolabelled p-mal- cMos. At the indicated times 15 μΐ of supernatant was analysed by PAGE-SDS and autoradiography. The gels were scanned by using a Typhoon™ Scanner and quantified using ImageJ software. Statistical analysis of the results obtained from two different independent experiments was performed by using an unpaired Student's t test. The percentatge of phosphorylated p-mal-cMos present at each time were expressed as mean ± S.D. Statistical differences between control versus either Arppl9 or a-Endosulfine in the two time-points were indicated. (* and ) p<0.03; (# and #) p< 0.04

Figure 4. Endogenous Arppl9 but not α-Endosulfine is required to promote correct entry into mitosis. (A) Interphase extracts were depleted of Cdc27 and either Arppl9 or a-Endosulfine and subsequently supplemented with cyclin A (final concentration 60 nM). (B) Mitotic extracts were depleted of Arppl9 or a- Endosulfme and the mitotic state was analysed. (C) 20 μΐ of interphase and mitotic egg extracts were immunoprecipitated with anti-C terminal a-Endosulfine or anti- human Arppl9 and the phosphorylation of these two proteins on the Gwl specific site was analysed. The staining signal was measured and quantified by using ImageJ software (D) Mitotic extracts were depleted of Cdc27 and Arppl9 and 20 minutes later were (+OA) or not (-OA) supplemented with Okadaic Acid (final concentration 0.7 μΜ) and the mitotic state was analysed. (E) HeLa cells were transfected or not (Lipo), with scramble (SC) or 50 or 100 nM of Arppl9 siRNA for 24 h, then synchronized by thymidine and released into nocodazole (50 ng/ml) for 10 h. The percentage of mitotic cells was measured by a 2D-FACS (propidium iodide/anti- phospho Serine Cdk antibody). The percentage of mitotic cells at each condition was expressed as mean ± S.D. Statistical differences between scramble versus either 50 nM or 100 nM Arppl9 siRNA are indicated. (*p <8.03E-06 and * p<! 45E--0 ). The cellular levels of endogenous Arppl9 at each condition are shown.

SUPPLEMENTARY FIGURE 1

Figure showing Xenopus Arppl9 and Xenopus α-Endosulfine protein sequence alignment (SEQID N° 1 and SEQ ID N° 2). Peptides identified by mass spectrometry are numbered from 1 to 4 and disposed over the corresponding sequence of Xenopus Arp l9. Neither of these peptides are specific to cc-Endosulfme, whereas three of them are specific of Xenopus Arppl9 protein.

SUPPLEMENTARY MATERIAL MATERIAL AND METHODS

Immunization procedures, protein purification and antibodies

Polyclonal phospho-tyrosine 15 Cdc2, anti-phospho-(Ser) CDK substrate and monoclonal PP2A/A (6G3) antibodies were obtained from Cell Signalling Technology Inc. Affinity purified antibodies against Gwl, Cdc27, cyclin A, cyclin B2 and Cdc25, were obtained as previously described (3-8). Monoclonal antibody anti-PP2A C subunit (1D6) was obtained from Upstate/Millipore. Phospho-S67/S62 Endo/Arpp antibody was obtained from Dr T. Hunt and Dr S Mochida.

Human pBKS-Arppl9 and pBKS-a-Endosulfme (obtained from Dr L. Gros) were subcloned into the EcoRl-Notl site of pGEX4T and into the EcoRl-Xhol site of pMal. The fusion proteins were expressed in Escherichia coli and the soluble fraction was purified using glutathione-sepharose and MBP-sepharose beads. Gst-human Arppl9 soluble protein was used to immunize rabbits. Immune sera was affinity purified on immobilized MBP-human Arppl9 column.

C-terminal-Xenopus α-Endosulfme antibodies were generated against the last 1 1 aminoacids of the C-terminal sequence of this protein. Peptides were coupled to thyroglobulin for immunization and to immobilized bovine serum albumin for affinity purification.

Preparation of Xenopus Egg Extracts

Mitotic egg extracts were prepared from unfertilized Xenopus eggs that were arrested at metaphase of the second meiotic division as previously described (9). Interphase egg extracts were prepared from de-jellied unfertilized eggs transferred in MMR/4 (25 mM NaCl, 0.5 mM KC1, 0.25 nM MgCl₂, 0.025 mM Na EGTA, 1.25 mM HEPES-NaOH ph 7.7) Extracts were prepared 40 minutes after ionophore addition by the same procedure as described for mitotic extracts.

HI Kinase Assays

Extract (1 μΐ) was frozen in liquid nitrogen at the indicated times. Samples were then thawed by the addition of l9 μΐ of HI buffer including [γ³³Ρ]ΑΤΡ and incubated for lO min at room temperature (10). Reactions were stopped by adding Laemmli sample buffer and analysed by SDS-PAGE.

Phosphatase activity

120 μΐ of interphase egg extracts were immunoprecipitated with 15 μΐ anti-PP2A/C monoclonal antibodies (1D6) bound to 120 μΐ of Dynabeads protein G magnetic beads. Immunoprecipitate was then split into four. One was not treated whereas the two others were incubated for 10 minutes at room temperature with 5.3 μg of either of thiophosphorylated Gst-Arppl9, Gst-a-Endosulfme or the a-Endosulfme mutant D66A. Radiolabeled p-mal-cMos was then added to each immunoprecipitated and incubated for the indicated times at 30°C in dephosphorylation buffer (50 mM Tris pH 8, 100 μΜ CaCl₂). To thiophosphorylate proteins, hyperactive Gwl was immunoprecipitated from 20 μΐ CSF translated extracts. The IP was subsequently washed three times with RIPA (NaH₂P0₄ lOmM, NaCl lOOmM, EDTA 5mM, Triton XI 00 1%, deoxycholate 0.5 %, β-glycerophosphate 80 μΜ, NaF 50 mM, DTT 1 mM), twice with Tris 50 mM ph 7.5 and subsequently used to phosphorylate 12.5 μg of each of these proteins of sample buffer containing ATP-5S (HEPES 20 mM, Mg Cl₂ 10 mM, ATP-5S 1 mM) at a final volume of 7 μΐ.

Biochemical purification

15 ml of interphase egg extracts were diluted with a volume of 55 ml of Tris 50 mM pH 7.5 and subsequently fractionated in a Heparine column. Elution was then precipitated with ammonium sulphate (cut off 50-70%), resuspended with 2 ml of Tris 50 mM pH 7.5 and dialysed over night against Tris 50 mM pH 7.5. The sample was then loaded in a Superdex Gel Filtration column (1.6 X 60 cm) and fractions of 2 ml corresponding to the molecular weight from 50 to 70 Kda and 20 to 50 Kda were recovered. 100 μΐ of these fractions were treated with FSBA at a final concentration of 40 μΜ to inhibit endogenous kinases and subsequently dialysed over night against Tris 50 mM pH 7.5 to remove free FSBA. Five microlitres of these two fractions were diluted with 3 μΐ of Tris 50 mM pH 7.5, 5 μΐ of phosphorylation buffer (HEPES 20 mM, MgCl₂ 10 mM, ATP 100 μΜ) and 2 μα of ΑΤΡγ³³ and were subsequently supplemented with hyperactive human Gwl (K72M) mutant immunoprecipitate. To obtain hyperactive human Gwl mutant immunoprecipitate, mitotic extracts were supplemented with the mRNA encoding the recombinant HA-human Gwl (K72M) mutant protein. After translation, the extracts were immunoprecipitated with anti- Gwl antibodies. An immunoprecipitate corresponding to 10 μΐ of Gwl-translated extract was used for each phosphorylation assay.

Mass spectrometry

Gel bands were submitted to tryptic digestion. The resultant peptides were analysed by MS and MS/ MS analysis with a W-TOF 2 hybrid quadruple/time-of-flight mass spectrometer (Micromass Ltd., Manchester, UK) equipped with a Z-spray ion source. LC/MS/MS data were processed automatically with the ProteinLynx Process (Micromass) module. Data analysis was performed with Mascot (Matrix Science Ltd., London, UK) against NCBI (The National Center for Biotechnology Information) database.

Gst-Pull Down

20 μΐ of Gst as a control, Gst-Arppl9, Gst-a-Endosulfme and Gst-Arppl9 mutant S62A, Gst--a-Endosulfme mutant S67A and Gst~a-Endosulfine mutant D66A bound to glutathione-sepharose beads were incubated with 20 μΐ of interphase or mitotic egg extracts. 20 minutes later the mix was centrifuged and the pellet was washed three times with RIP A and three times with Tris 50 mM pH 7.5. An aliquot of 1.1 μΐ of interphase extracts as well as the different pellets were then analysed by western blot.

siRNA design and transfection

The promega T7 Ribomax Express RNAi system was used to produce in vitro transcribed siRNAs against human Arppl9. Transfection of siRNA was performed using Lipofectamine RNAiMax Reagent (Initrogen) as per the manufacture's instructions. Briefly, cells were transfected with the corresponding siRNA for 24 hours and subsequently synchronized with thymidine (2.5 mM) a subsequent period of 24 hours. Finally cells were released into 50 ng/ml of Nododazole and analysed by 2D-FACS (propiddium iodide and anti-phospho-(Ser) Cdk substrate antibody) to determinate the percentage of mitotic cells. References

1. V. Archambault, X. Zhao, H. White-Cooper, A. T. Carpenter, D. M. Glover, PLoS Genet 3, e200 (Nov, 2007).

2. D. Bataille et al, Cell Mol Life Sci 56, 78 (Oct 1, 1999).

3. T. Lorca et al, J Cell Sci 123, 2281 (Jul 1).

4. A. Abrieu et al , J Cell Sci 111 ( Pt 12), 1751 (Jun, 1998).

5. C. Bernis et al, EMBO Rep 8, 91 (Jan, 2007).

6. A. Burgess et al, Proc Natl Acad Sci USA 107, 12564 (Jul 13).

7. T. Lorca et al, Embo J Xl, 3565 (1998).

8. S. Vigneron et al, EMBO J 2$, 2786 (Sep 16, 2009).

9. A. Castro et al., Mol Biol Cell 12, 2660 (2001).

10. T. Lorca et al, Nature 366, 270 (Nov 18, 1993).

Claims

1. Arp l9 and/or a-Endosulfine for use as specific inhibitors of the cell cycle deregulation.

2. Arppl9 and/or α-Endosulfine for use as specific inhibitors of PP2A.

3. Arppl9 and/or α-Endosulfine for use as specific inhibitors of PP2A Β55δ.

4. Pharmaceutical compositions comprising an efficient amount of Arppl9 and α-Endosulfine in association with pharmaceutically acceptable carriers.